PROTEIN EXPRESSION SYSTEMS

摘要

The invention is based on an expression enhancer sequence derived from the RNA-2 genome segment of a bipartite RNA virus, in which a target initiation site in the RNA-2 genome segment has been mutated. Deletion of appropriate start codons upstream of the main RNA2 translation initiation can greatly increase in foreign protein accumulation without the need for viral replication. Also provided are methods, vectors and systems, including the ‘hyper-translatable’ Cowpea Mosaic Virus (“CPMV-HT”) based protein expression system.

主权项

1. A gene expression construct comprising:
(a) an expression enhancer sequence derived from the RNA-2 genome segment of a Comoviridae bipartite RNA virus, in which a target initiation site in the RNA-2 genome segment has been mutated,
wherein the wild-type RNA2 genome segment of the Comoviridae virus encodes two carboxy coterminal proteins through two different translation initiation sites located in the same triplet reading frame, and wherein the mutated initiation site is the first of these two initiation sites and thus corresponds to the initiation site at position 161 in the wild-type RNA-2 segment of cowpea mosaic virus (CPMV),wherein the expression enhancer sequence comprises at least a 100 nucleotide sequence of the comoviral RNA-2 genome segment which includes the mutated initiation site; and (b) a heterologous sequence for facilitating insertion of a gene encoding a protein of interest into the gene expression construct, wherein the heterologous sequence is located downstream of the mutated target initiation site in the enhancer sequence; and optionally (c) a 3′ UTR.